It has long been known that skeletal muscle twitch contraction persists in the absence of extracellular Ca2+. Nevertheless, recent studies, including our own, have identified a rapidly activated store-operated Ca2+ entry (SOCE) pathway in skeletal muscle used to replenish previously depleted intracellular Ca2+ stores. We provide strong preliminary data to demonstrate that STIM1 proteins function as the calcium sensor of the sarcoplasmic reticulum (SR) and Orai1 proteins as the calcium permeable sarcolemmal channel in adult skeletal muscle fibers. In addition, STIM1 and Orai1 proteins are known to be expressed at high levels in skeletal muscle and a debilitating myopathy is consistently observed in human severe combined immuno-deficiency patients possessing either loss-of-function STIM1 or Orai1 mutations. Together, these results demonstrate that SOCE likely plays a previously unappreciated role in skeletal muscle function and disease; indicating that STIM1- Orai1 coupling represents an exciting and untapped frontier in skeletal muscle Ca2+ signaling. Based on these findings, and by analogy to SOCE in non-excitable cells, we hypothesize that STIM1 and Orai1 proteins pre-localize to the triad junction to coordinate uniquely rapid SOCE activation that limits SR Ca2+ store depletion during repetitive tetanic stimulation. Chronic SOCE contributes to muscle fiber hyperexcitability and deterioration in a mouse model of malignant hyperthermia (MH) and central core disease (CCD). We will test the validity of this hypothesis by characterizing the: 1) molecular mechanism of rapid SOCE activation in adult skeletal muscle (Aim 1), 2) role of SOCE in maintaining SR Ca2+ content and release during repetitive high frequency tetanic stimulation (Aim 2), and 3) degree to which SOCE dysfunction contributes/modifies muscle disease. An important conceptual innovation of this project is the hypothesis that a SOCE activity limits activity-dependent skeletal muscle performance and serves as an important modifier of muscle disease. An important technological innovation is our recent development of skeletal muscle-specific dominant negative Orai1 transgenic mice and use of these mice to probe the (patho)physiological role of SOCE in skeletal muscle. This project will employ a battery of molecular/cell biological, bi-molecular fluorescence complementation, fluorescence energy resonance transfer, electrophysiological, confocal imaging, biochemical, transgenic, electron microscopy, in vitro muscle contraction, and in vivo muscle performance assays to characterize the mechanism of STIM1-Orai1 activation and the functional role of SOCE in skeletal muscle. The molecular mechanisms characterized during this venture will have implications not only for muscle function, but will also extend more broadly to disorders of Ca2+ dysregulation across a diversity of related clinical arenas including immunodeficiency, autoimmune disease, fatigue, and myopathy. Thus, discoveries resulting from this proposal will provide promise for the development of novel approaches, treatments, and diagnostics for a wide range of diseases in Ca2+ signaling.